Colorectal cancer (CRC) is the third leading cause of cancer death in the US. It is difficult to cure CRC because majority of the existing therapies fail to significantly obliterate seeds of cancer called `cancer stem cells (CRCSCs). Cellular cholesterol metabolism is a single most important target for CRCSCs. However, existing therapies that target this pathway, e.g. statins fail to significantly inhibit cholesterol levels in the cells due to redundant mechanisms that govern cholesterol levels in cancer cells. However, cholesterol utilization is regulated by select group of specific cholesterol transport proteins, which if targeted, can have critical effect on plasma membrane (PM) biophysical properties of CRC cells. We have identified a cholesterol transport protein called StarD5, which is the only known mammalian protein that regulates cholesterol transport to cell membrane. StarD5 is significantly overexpressed in human colon cancer tissues and particularly in colon CRCSCs. Inhibition of StarD5 resulted in significant inhibition of colon CSCs in vitro and in vivo, as well as cholesterol contents in PM resulting in increased PM fluidity and robust inhibition of Insulin-like growth factor-1 receptor (IGF1R) and epidermal growth factor receptor (EGFR) signaling. We propose to examine, in detail, the significance of StarD5 overexpression on colon cancer initiation, progression, and patient outcomes as well as impact of StarD5 modulation on CRCSC phenotype in human CRCSCs (Aim 1). We would also like to understand mechanisms of how StarD5 inhibition regulates CSCs. To that end, we propose to determine the effect of StarD5 inhibition on PM fluidity, influx of platinum agents, and growth factor signaling initiated in PM (Aim 2). Moreover, we plan to examine the efficacy of small molecular inhibitors of StarD5 (SD5i) on CRCSC phenotype using primary human colon spheroids as well as determine their efficacy of in combination with FDA-approved chemotherapy and targeted therapy (panitumumab) against advanced animal models of CSCs (Aim 3). The studies will provide novel insight into how alternation in membrane cholesterol regulates cancer growth, and provide a novel class of target for cancer therapies that may lead to long-term remission and/or cure by targeting therapy resistant CRCSCs.